43
with 1x TBS-T. The ECL solution (see below) was prepared during this washing step.
For one membrane, 2.5 mL of each fresh ECL solution were mixed and the mixture was pipetted onto the membrane. After 2 min incubation, the membrane was visualized withGene Gnome. The exposure times of each membrane differed from 5 x 2 min to 5 x 3 min depending on the signal strength.
1x PBS (2000 mL)
Na2HPO4 2.3 g
KH2PO4 0.5 g
NaCl 18.0 g
deionized water add to 2000 mL
KH2PO4 dissolves at last, adjust to pH 7.4 with NaOH before making up to 2000 mL
1x PBS-T (1000 mL)
1x PBS 999 mL
Tween 20 1 mL
1x TBS-T (2000 mL)
Tris 4.84 g
NaCl 17.52 g
Tween 20 2 mL
deionized water add to 2000 mL
ECL solution (10 mL)
solution A 250 mM luminol (44.29 g/L) 100 µL 90 mM p-coumaric acid (14.77 g/L) 44 µL 1 M Tris/HCl, pH 8.5 1000 µL
deionized water 8856 µL
solution B 30% H2O2 (w/v) 6.1 µL 1 M Tris/HCl, pH 8.5 1000 µL
deionized water 8993.9 µL
mix solution A and B (1:1) shortly before use
3.6.5 Competitive inhibition
The specificity of the staining with anti-mTff1-1 and anti-rTff3-2, respectively, was tested by competition with the corresponding synthetic peptides (see chapter 2.3.3).
One mL antiserum (anti-mTff1-1 or anti-rTff3-2, diluted in 1x TBS-T buffer, 1:500) was pre-adsorbed with 10 µg corresponding synthetic peptide by shaking at 4°C overnight and then used for Western blot analysis.
min each at room temperature, and then incubated with the secondary antibody, Cy3-labeled anti-rabbit-IgG F(ab’)2 fragment from sheep antiserum (see chapter 2.3.3), 1:200 diluted in blocking buffer at room temperature for 1 h (from this step, the incubation and all following steps were carried out in the dark) and rinsed again with PBS/pH 7.4 2 times 5 min each at room temperature.
Blocking buffer (10 mL)
goat serum 1 mL (final conc. 10%)
10% NaN3 100 µL (final conc. 0.1%)
10% Triton™ X-100 300 µL (final conc. 0.3%)
1x PBS/pH 7.4 add to 10 mL
The cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole dihydrochloride) at room temperature for 5 min using a 1:1000 diluted DAPI stock solution (0.1 mg/mL in PBS/pH 7.4). Subsequently, the cover slips were rinsed twice with PBS/pH 7.4 at room temperature for 5 min and once with deionized water for 5 min. The cover slips were embedded on the microscope slides using DAKO fluorescent mounting medium.
The slides were photographed with an Axiophot microscope equipped with an AxioCam digital camera. The following filter sets were used: filterset 01 for DAPI and filterset 15 for Cy3 (see chapter 2.7). The images were processed with the AxioVision 3.1 software.
3.7.1.2 Primary rat brain cells
All procedures were performed at room temperature. After 20–22 DIV (days in vitro), primary culture cells (provided from Dr. Stellmacher, IPT, Magdeburg) grown on glass cover slips were fixed with periodate-lysine-paraformaldehyde (PLP) fix (see below) for 30 min. Cells were washed with PBS 3 times and blocked with blocking solution (see below) for 1 h. Subsequently, cells were incubated with primary antibody (see chapter 2.3.2) diluted in blocking solution for 1 h. After 3 times washing with PBS, cells were incubated with secondary antibodies (see chapter 2.3.3) diluted in block solution for 1 h.
Cells were incubated with DAPI for 3 min to stain the cell nuclei. Subsequently, cells were washed 2 times with PBS, once with water, and mounted with Mowiol 4-88 onto the slide. The following antibodies were used (see also in chapter 2.3): primary antibodies: goat anti-Iba-1, rabbit anti-Iba-1, chicken anti-GFAP, mouse anti-MAP2, and rabbit anti-rTff3-2 antiserum; secondary antibodies: donkey anti-goat Cy3, Alexa Fluor™ 488 goat anti–rabbit, donkey anti-chicken Cy3, DyLight™ 649 donkey anti–chicken, donkey anti-mouse Cy3, goat anti-rabbit Cy3.
PLP fix
PFA (paraformaldehyde) 4%
sucrose 5.4%
NaIO4 0.01 M
lysine HCl 0.1M
dissolved in 0.1M sodium phosphate buffer/pH 7.4
Blocking solution
horse serum 10%
sucrose 5%
BSA 0.01 M
saponin 0.2 mg/mL
dissolved in PBS buffer, pH 7.4
45
Cells were viewed and recorded digitally using the Zeiss microscope Axio Observer.Z1 equipped with a camera AxioCam MRm and the AxioVision Release 4.8 software. The excitation and emission spectra of the fluorescent markers for all the immunofluorescence staining are listed below. Images were processed using Adobe Photoshop 7.0.
Excitation and emission spectra of the fluorescent markers
Fluorescence Marker Excitation (nm) Emission (nm) Colour/Spectrum
Alexa Fluor 488 499 519 green
Cy3 552 565 red
Alexa Fluor 647 652 668 infrared
DyLight 649 654 673 infrared
DAPI 345 455 blue
3.7.1.3 Quantitative analysis
Photos of the RGM-1 cells after in vitro wounding were captured in such way that both stationary and migratory cell regions were visible. All photos had the same frame size.
For each photo, the total Tff1 immunofluorescence signal was analyzed within a normalized area of stationary and migratory cells, respectively, with GeneTools (Figure 13). This signal was divided by the number of cells in order to obtain the Tff1 staining per cell. All data were then analyzed withExcel 2003.
Figure 13: Schematic presentation of total Tff1 immunofluorescence signal analysis The rectangular standardized zones (stationary (s) and migratory (m)) were marked by a red line.
The scratch wound is indicated by the white dotted line. (A) Using GeneTools software, total Tff1 immunofluorescence was measured within a pair of rectangular standardized zones (s and m).
(B) The cell number within the corresponding areas was counted after staining the nuclei with DAPI. Scale bar: 20µm.
3.7.2 Tissues
3.7.2.1 Tissue preparation
Mouse brains were carefully dissected (see chapter 3.3.3). The right half used for freeze sections was immediately fixed in 4% PFA (in 50 mM HEPES-Puffer/pH 7.4) overnight at 4°C, and then transferred to 1 M sucrose (dissolved in 1x PBS/pH 7.4) overnight at 4°C. Subsequently, the tissues were embedded in OCT compound (Tissue-Tek®), frozen in 2-methylbutane (pre-frozen at -60°C) for 2 min, and then stored at -80°C. To prepare sagittal sections for free floating immunohistochemical staining, the frozen half hemisphere was cut using Leica Jung CM3000 Cryostat Microtome at -20°C. The 25 µm sagittal sections were cut and carefully collected into
24-well plate containing 1x PBS and used for further experiments. For long-term storage, sections were frozen in 1 M sucrose at -20°C.
3.7.2.2 Free floating immunohistochemistry
Free-floating sections were washed 3 times for 10 min each with PBS/pH 7.4 at room temperature. Sections were then incubated in 50% (v/v) methanol solution in 1x PBS/pH 7.4 with 1% (v/v) H2O2 (fresh made) at room temperature for 10 min to block the endogenous peroxidase activity. Then sections were washed again 3 times for 10 min each with PBS/pH 7.4 at room temperature. The non-specific background staining of sections were blocked with freshly made blocking buffer I (see below) at room temperature for 60 min. Then, sections were incubated with the primary antibodies mTff1-1 and Iba-1 (see chapter 2.3.2), diluted in blocking buffer I, for 1-3 days at 4°C.
Sections were then washed 3 times for 10 min each with PBS/pH 7.4 at room temperature and blocked again with blocking buffer II (see below) for 60 min at room temperature. Sections were incubated with the secondary antibody (biotinylated goat anti-rabbit IgG antibody, see chapter 2.3.3) diluted in blocking buffer II at room temperature overnight. After that, sections were washed 3 times for 10 min each with PBS/pH 7.4 at room temperature.
The avidin-biotin peroxidase complex (ABC) method (VECTASTAIN® Elite ABC Kit, BIOZOL GmbH) was used following the manufacturer's instructions. First, sections were blocked with 1x BSA (0.2%) solution at room temperature for 60 min. During this time, solution A and solution B from the VECTASTAIN® Elite ABC Kit were both 1:1000 diluted in 1x BSA solution (see below) and incubated at room temperature for 30 min.
After the blocking, sections were further incubated with this mixture, overnight at 4°C or more than 4 h at room temperature. Then, sections were washed two times with 1x PBS/pH 7.4 and once with 1x Tris-buffer/pH 7.6 (0.05 M) for 10 min each at room temperature. During this washing step, 0.5 g/L DAB (diaminobenzidine) solution (in 1x Tris-buffer/pH 7.6) as chromogen was freshly made and 0.5 mL DAB solution was added to each well of the 24-well plate containing sections. To start the staining reaction, 20 µL 3% H2O2 was added to each well and incubated for 2-4 minutes under visual control to avoid strong background. The reaction was stopped with PBS washing for three times and then mounted onto gelatine-coated slides (pretreated with 0.5%
gelatine; air dried and stored at 4°C). After being air-dried, the slides with mounted sections were progressively dehydrated: first with series of ethanol (concentration elevated from 70% to 100%), 2 min each; then with xylol (two times, for 2 min and 5 min). At the end, cover slips were mounted with DPX mounting medium on the slides.
After air drying overnight, the slides were examined under the microscope BZ-8000 (Keyence) and then processed with the BZ observation application software (Keyence).
Blocking buffer I for immunohistochemical staining (10 mL)
goat serum 1 mL (final conc. 10%)
10% NaN3 100 µL (final conc. 0.1%)
10% Triton™ X-100 300 µL (final conc. 0.3%)
1x PBS/pH 7.4 add to 10 mL
47 Blocking buffer II for immunohistochemical staining (10 mL)
goat serum 1 mL (final conc. 10%)
10% NaN3 100 µL (final conc. 0.1%)
10% Triton™ X-100 100 µL (final conc. 0.1%)
1x PBS/pH 7.4 add to 10 mL
Mixture from VECTASTAIN® Elite ABC Kit (10 mL)
2% BSA 1 mL (final conc. 0.2%)
solution A from ABC Kit 10 µL
solution B from ABC Kit 10 µL
1x PBS/pH 7.4 add to 10 mL
3.7.3 Competitive inhibition
The specificity of the staining with anti-mTff1-1 and anti-rTff3-2, respectively, was tested by competition with the corresponding synthetic peptides, i.e., 1 mL antiserum (anti-mTff1-1 or anti-rTff3-2, diluted in blocking buffer) was pre-adsorbed with 10 µg corresponding synthetic peptide by shaking at 4°C overnight and then used for immunohistochemistry.